Genetic and hypoxic alterations of the micro <scp>RNA</scp> ‐210‐ <scp>ISCU</scp> 1/2 axis promote iron–sulfur deficiency and pulmonary hypertension

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Research Article30 March 2015Open Access Source Data Genetic and hypoxic alterations of the microRNA-210-ISCU1/2 axis promote iron–sulfur deficiency and pulmonary hypertension Kevin White Kevin White Divisions of Cardiovascular Medicine and Network Medicine, Department of Medicine, Brigham and Women's Hospital, Harvard Medical School, Boston, MA, USA Search for more papers by this author Yu Lu Yu Lu Divisions of Cardiovascular Medicine and Network Medicine, Department of Medicine, Brigham and Women's Hospital, Harvard Medical School, Boston, MA, USA Search for more papers by this author Sofia Annis Sofia Annis Divisions of Cardiovascular Medicine and Network Medicine, Department of Medicine, Brigham and Women's Hospital, Harvard Medical School, Boston, MA, USA Search for more papers by this author Andrew E Hale Andrew E Hale Divisions of Cardiovascular Medicine and Network Medicine, Department of Medicine, Brigham and Women's Hospital, Harvard Medical School, Boston, MA, USA Search for more papers by this author B Nelson Chau B Nelson Chau Regulus Therapeutics, San Diego, CA, USA Search for more papers by this author James E Dahlman James E Dahlman Institute for Medical Engineering and Science, Massachusetts Institute of Technology, Cambridge, MA, USA Harvard-MIT Division of Health Sciences and Technology, Massachusetts Institute of Technology, Cambridge, MA, USA Department of Chemical Engineering, Massachusetts Institute of Technology, Cambridge, MA, USA David H. Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, Cambridge, MA, USA Search for more papers by this author Craig Hemann Craig Hemann The Davis Heart and Lung Research Institute, Division of Cardiovascular Medicine, Department of Internal Medicine, Wexner Medical Center, The Ohio State University, Columbus, OH, USA Search for more papers by this author Alexander R Opotowsky Alexander R Opotowsky Divisions of Cardiovascular Medicine and Network Medicine, Department of Medicine, Brigham and Women's Hospital, Harvard Medical School, Boston, MA, USA Department of Cardiology, Boston Children's Hospital, Harvard Medical School, Boston, MA, USA Search for more papers by this author Sara O Vargas Sara O Vargas Department of Pathology, Boston Children's Hospital, Harvard Medical School, Boston, MA, USA Search for more papers by this author Ivan Rosas Ivan Rosas Division of Pulmonary/Critical Care Medicine, Department of Medicine, Harvard Medical School, Boston, MA, USA Search for more papers by this author Mark A Perrella Mark A Perrella Division of Pulmonary/Critical Care Medicine, Department of Medicine, Harvard Medical School, Boston, MA, USA Department of Pediatric Newborn Medicine, Brigham and Women's Hospital, Harvard Medical School, Boston, MA, USA Search for more papers by this author Juan C Osorio Juan C Osorio Division of Pulmonary/Critical Care Medicine, Department of Medicine, Harvard Medical School, Boston, MA, USA Search for more papers by this author Kathleen J Haley Kathleen J Haley Division of Pulmonary/Critical Care Medicine, Department of Medicine, Harvard Medical School, Boston, MA, USA Search for more papers by this author Brian B Graham Brian B Graham Program in Translational Lung Research, University of Colorado, Denver, Aurora, CO, USA Search for more papers by this author Rahul Kumar Rahul Kumar Program in Translational Lung Research, University of Colorado, Denver, Aurora, CO, USA Search for more papers by this author Rajan Saggar Rajan Saggar Departments of Medicine and Pathology, David Geffen School of Medicine, University of California, Los Angeles, Los Angeles, CA, USA Search for more papers by this author Rajeev Saggar Rajeev Saggar Department of Cardiothoracic Surgery, University of Arizona College of Medicine, Phoenix, AZ, USA Search for more papers by this author W Dean Wallace W Dean Wallace Departments of Medicine and Pathology, David Geffen School of Medicine, University of California, Los Angeles, Los Angeles, CA, USA Search for more papers by this author David J Ross David J Ross Departments of Medicine and Pathology, David Geffen School of Medicine, University of California, Los Angeles, Los Angeles, CA, USA Search for more papers by this author Omar F Khan Omar F Khan Department of Chemical Engineering, Massachusetts Institute of Technology, Cambridge, MA, USA David H. Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, Cambridge, MA, USA Search for more papers by this author Andrew Bader Andrew Bader Institute for Medical Engineering and Science, Massachusetts Institute of Technology, Cambridge, MA, USA Harvard-MIT Division of Health Sciences and Technology, Massachusetts Institute of Technology, Cambridge, MA, USA Search for more papers by this author Bernadette R Gochuico Bernadette R Gochuico Medical Genetics Branch, National Human Genome Research Institute, National Institutes of Health, Bethesda, MD, USA Search for more papers by this author Majed Matar Majed Matar Celsion-EGEN, Inc., Huntsville, AL, USA Search for more papers by this author Kevin Polach Kevin Polach Celsion-EGEN, Inc., Huntsville, AL, USA Search for more papers by this author Nicolai M Johannessen Nicolai M Johannessen Department of Cardiology, University of Bergen, Bergen, Norway Search for more papers by this author Haydn M Prosser Haydn M Prosser The Wellcome Trust Sanger Institute, Wellcome Trust Genome Campus, Hinxton, Cambridge, UK Search for more papers by this author Daniel G Anderson Daniel G Anderson Institute for Medical Engineering and Science, Massachusetts Institute of Technology, Cambridge, MA, USA Harvard-MIT Division of Health Sciences and Technology, Massachusetts Institute of Technology, Cambridge, MA, USA Department of Chemical Engineering, Massachusetts Institute of Technology, Cambridge, MA, USA David H. Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, Cambridge, MA, USA Search for more papers by this author Robert Langer Robert Langer Institute for Medical Engineering and Science, Massachusetts Institute of Technology, Cambridge, MA, USA Harvard-MIT Division of Health Sciences and Technology, Massachusetts Institute of Technology, Cambridge, MA, USA Department of Chemical Engineering, Massachusetts Institute of Technology, Cambridge, MA, USA David H. Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, Cambridge, MA, USA Search for more papers by this author Jay L Zweier Jay L Zweier The Davis Heart and Lung Research Institute, Division of Cardiovascular Medicine, Department of Internal Medicine, Wexner Medical Center, The Ohio State University, Columbus, OH, USA Search for more papers by this author Laurence A Bindoff Laurence A Bindoff Department of Clinical Medicine, University of Bergen, Bergen, Norway Department of Neurology, Haukeland University Hospital, Bergen, Norway Search for more papers by this author David Systrom David Systrom Division of Pulmonary/Critical Care Medicine, Department of Medicine, Harvard Medical School, Boston, MA, USA Search for more papers by this author Aaron B Waxman Aaron B Waxman Division of Pulmonary/Critical Care Medicine, Department of Medicine, Harvard Medical School, Boston, MA, USA Search for more papers by this author Richard C Jin Richard C Jin Divisions of Cardiovascular Medicine and Network Medicine, Department of Medicine, Brigham and Women's Hospital, Harvard Medical School, Boston, MA, USA Search for more papers by this author Stephen Y Chan Corresponding Author Stephen Y Chan Divisions of Cardiovascular Medicine and Network Medicine, Department of Medicine, Brigham and Women's Hospital, Harvard Medical School, Boston, MA, USA Search for more papers by this author Kevin White Kevin White Divisions of Cardiovascular Medicine and Network Medicine, Department of Medicine, Brigham and Women's Hospital, Harvard Medical School, Boston, MA, USA Search for more papers by this author Yu Lu Yu Lu Divisions of Cardiovascular Medicine and Network Medicine, Department of Medicine, Brigham and Women's Hospital, Harvard Medical School, Boston, MA, USA Search for more papers by this author Sofia Annis Sofia Annis Divisions of Cardiovascular Medicine and Network Medicine, Department of Medicine, Brigham and Women's Hospital, Harvard Medical School, Boston, MA, USA Search for more papers by this author Andrew E Hale Andrew E Hale Divisions of Cardiovascular Medicine and Network Medicine, Department of Medicine, Brigham and Women's Hospital, Harvard Medical School, Boston, MA, USA Search for more papers by this author B Nelson Chau B Nelson Chau Regulus Therapeutics, San Diego, CA, USA Search for more papers by this author James E Dahlman James E Dahlman Institute for Medical Engineering and Science, Massachusetts Institute of Technology, Cambridge, MA, USA Harvard-MIT Division of Health Sciences and Technology, Massachusetts Institute of Technology, Cambridge, MA, USA Department of Chemical Engineering, Massachusetts Institute of Technology, Cambridge, MA, USA David H. Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, Cambridge, MA, USA Search for more papers by this author Craig Hemann Craig Hemann The Davis Heart and Lung Research Institute, Division of Cardiovascular Medicine, Department of Internal Medicine, Wexner Medical Center, The Ohio State University, Columbus, OH, USA Search for more papers by this author Alexander R Opotowsky Alexander R Opotowsky Divisions of Cardiovascular Medicine and Network Medicine, Department of Medicine, Brigham and Women's Hospital, Harvard Medical School, Boston, MA, USA Department of Cardiology, Boston Children's Hospital, Harvard Medical School, Boston, MA, USA Search for more papers by this author Sara O Vargas Sara O Vargas Department of Pathology, Boston Children's Hospital, Harvard Medical School, Boston, MA, USA Search for more papers by this author Ivan Rosas Ivan Rosas Division of Pulmonary/Critical Care Medicine, Department of Medicine, Harvard Medical School, Boston, MA, USA Search for more papers by this author Mark A Perrella Mark A Perrella Division of Pulmonary/Critical Care Medicine, Department of Medicine, Harvard Medical School, Boston, MA, USA Department of Pediatric Newborn Medicine, Brigham and Women's Hospital, Harvard Medical School, Boston, MA, USA Search for more papers by this author Juan C Osorio Juan C Osorio Division of Pulmonary/Critical Care Medicine, Department of Medicine, Harvard Medical School, Boston, MA, USA Search for more papers by this author Kathleen J Haley Kathleen J Haley Division of Pulmonary/Critical Care Medicine, Department of Medicine, Harvard Medical School, Boston, MA, USA Search for more papers by this author Brian B Graham Brian B Graham Program in Translational Lung Research, University of Colorado, Denver, Aurora, CO, USA Search for more papers by this author Rahul Kumar Rahul Kumar Program in Translational Lung Research, University of Colorado, Denver, Aurora, CO, USA Search for more papers by this author Rajan Saggar Rajan Saggar Departments of Medicine and Pathology, David Geffen School of Medicine, University of California, Los Angeles, Los Angeles, CA, USA Search for more papers by this author Rajeev Saggar Rajeev Saggar Department of Cardiothoracic Surgery, University of Arizona College of Medicine, Phoenix, AZ, USA Search for more papers by this author W Dean Wallace W Dean Wallace Departments of Medicine and Pathology, David Geffen School of Medicine, University of California, Los Angeles, Los Angeles, CA, USA Search for more papers by this author David J Ross David J Ross Departments of Medicine and Pathology, David Geffen School of Medicine, University of California, Los Angeles, Los Angeles, CA, USA Search for more papers by this author Omar F Khan Omar F Khan Department of Chemical Engineering, Massachusetts Institute of Technology, Cambridge, MA, USA David H. Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, Cambridge, MA, USA Search for more papers by this author Andrew Bader Andrew Bader Institute for Medical Engineering and Science, Massachusetts Institute of Technology, Cambridge, MA, USA Harvard-MIT Division of Health Sciences and Technology, Massachusetts Institute of Technology, Cambridge, MA, USA Search for more papers by this author Bernadette R Gochuico Bernadette R Gochuico Medical Genetics Branch, National Human Genome Research Institute, National Institutes of Health, Bethesda, MD, USA Search for more papers by this author Majed Matar Majed Matar Celsion-EGEN, Inc., Huntsville, AL, USA Search for more papers by this author Kevin Polach Kevin Polach Celsion-EGEN, Inc., Huntsville, AL, USA Search for more papers by this author Nicolai M Johannessen Nicolai M Johannessen Department of Cardiology, University of Bergen, Bergen, Norway Search for more papers by this author Haydn M Prosser Haydn M Prosser The Wellcome Trust Sanger Institute, Wellcome Trust Genome Campus, Hinxton, Cambridge, UK Search for more papers by this author Daniel G Anderson Daniel G Anderson Institute for Medical Engineering and Science, Massachusetts Institute of Technology, Cambridge, MA, USA Harvard-MIT Division of Health Sciences and Technology, Massachusetts Institute of Technology, Cambridge, MA, USA Department of Chemical Engineering, Massachusetts Institute of Technology, Cambridge, MA, USA David H. Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, Cambridge, MA, USA Search for more papers by this author Robert Langer Robert Langer Institute for Medical Engineering and Science, Massachusetts Institute of Technology, Cambridge, MA, USA Harvard-MIT Division of Health Sciences and Technology, Massachusetts Institute of Technology, Cambridge, MA, USA Department of Chemical Engineering, Massachusetts Institute of Technology, Cambridge, MA, USA David H. Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, Cambridge, MA, USA Search for more papers by this author Jay L Zweier Jay L Zweier The Davis Heart and Lung Research Institute, Division of Cardiovascular Medicine, Department of Internal Medicine, Wexner Medical Center, The Ohio State University, Columbus, OH, USA Search for more papers by this author Laurence A Bindoff Laurence A Bindoff Department of Clinical Medicine, University of Bergen, Bergen, Norway Department of Neurology, Haukeland University Hospital, Bergen, Norway Search for more papers by this author David Systrom David Systrom Division of Pulmonary/Critical Care Medicine, Department of Medicine, Harvard Medical School, Boston, MA, USA Search for more papers by this author Aaron B Waxman Aaron B Waxman Division of Pulmonary/Critical Care Medicine, Department of Medicine, Harvard Medical School, Boston, MA, USA Search for more papers by this author Richard C Jin Richard C Jin Divisions of Cardiovascular Medicine and Network Medicine, Department of Medicine, Brigham and Women's Hospital, Harvard Medical School, Boston, MA, USA Search for more papers by this author Stephen Y Chan Corresponding Author Stephen Y Chan Divisions of Cardiovascular Medicine and Network Medicine, Department of Medicine, Brigham and Women's Hospital, Harvard Medical School, Boston, MA, USA Search for more papers by this author Author Information Kevin White1,21,‡, Yu Lu1,‡, Sofia Annis1, Andrew E Hale1, B Nelson Chau2,22, James E Dahlman3,4,5,6, Craig Hemann7, Alexander R Opotowsky1,8, Sara O Vargas9, Ivan Rosas10, Mark A Perrella10,11, Juan C Osorio10, Kathleen J Haley10, Brian B Graham12, Rahul Kumar12, Rajan Saggar13, Rajeev Saggar14, W Dean Wallace13, David J Ross13, Omar F Khan5,6, Andrew Bader3,4, Bernadette R Gochuico15, Majed Matar16, Kevin Polach16, Nicolai M Johannessen17, Haydn M Prosser18, Daniel G Anderson3,4,5,6, Robert Langer3,4,5,6, Jay L Zweier7, Laurence A Bindoff19,20, David Systrom10, Aaron B Waxman10, Richard C Jin1 and Stephen Y Chan 1 1Divisions of Cardiovascular Medicine and Network Medicine, Department of Medicine, Brigham and Women's Hospital, Harvard Medical School, Boston, MA, USA 2Regulus Therapeutics, San Diego, CA, USA 3Institute for Medical Engineering and Science, Massachusetts Institute of Technology, Cambridge, MA, USA 4Harvard-MIT Division of Health Sciences and Technology, Massachusetts Institute of Technology, Cambridge, MA, USA 5Department of Chemical Engineering, Massachusetts Institute of Technology, Cambridge, MA, USA 6David H. Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, Cambridge, MA, USA 7The Davis Heart and Lung Research Institute, Division of Cardiovascular Medicine, Department of Internal Medicine, Wexner Medical Center, The Ohio State University, Columbus, OH, USA 8Department of Cardiology, Boston Children's Hospital, Harvard Medical School, Boston, MA, USA 9Department of Pathology, Boston Children's Hospital, Harvard Medical School, Boston, MA, USA 10Division of Pulmonary/Critical Care Medicine, Department of Medicine, Harvard Medical School, Boston, MA, USA 11Department of Pediatric Newborn Medicine, Brigham and Women's Hospital, Harvard Medical School, Boston, MA, USA 12Program in Translational Lung Research, University of Colorado, Denver, Aurora, CO, USA 13Departments of Medicine and Pathology, David Geffen School of Medicine, University of California, Los Angeles, Los Angeles, CA, USA 14Department of Cardiothoracic Surgery, University of Arizona College of Medicine, Phoenix, AZ, USA 15Medical Genetics Branch, National Human Genome Research Institute, National Institutes of Health, Bethesda, MD, USA 16Celsion-EGEN, Inc., Huntsville, AL, USA 17Department of Cardiology, University of Bergen, Bergen, Norway 18The Wellcome Trust Sanger Institute, Wellcome Trust Genome Campus, Hinxton, Cambridge, UK 19Department of Clinical Medicine, University of Bergen, Bergen, Norway 20Department of Neurology, Haukeland University Hospital, Bergen, Norway 21Present address: Novartis, Cambridge, MA, USA 22Present address: RaNA Therapeutics, Cambridge, MA, USA ‡These authors contributed equally to this work *Corresponding author. Tel: +1 617 525 4844; Fax: +1 617 525 4830; E-mail: [email protected] EMBO Mol Med (2015)7:695-713https://doi.org/10.15252/emmm.201404511 See also: H Tang et al (June 2015) PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions Figures & Info Abstract Iron–sulfur (Fe-S) clusters are essential for mitochondrial metabolism, but their regulation in pulmonary hypertension (PH) remains enigmatic. We demonstrate that alterations of the miR-210-ISCU1/2 axis cause Fe-S deficiencies in vivo and promote PH. In pulmonary vascular cells and particularly endothelium, hypoxic induction of miR-210 and repression of the miR-210 targets ISCU1/2 down-regulated Fe-S levels. In mouse and human vascular and endothelial tissue affected by PH, miR-210 was elevated accompanied by decreased ISCU1/2 and Fe-S integrity. In mice, miR-210 repressed ISCU1/2 and promoted PH. Mice deficient in miR-210, via genetic/pharmacologic means or via an endothelial-specific manner, displayed increased ISCU1/2 and were resistant to Fe-S-dependent pathophenotypes and PH. Similar to hypoxia or miR-210 overexpression, ISCU1/2 knockdown also promoted PH. Finally, cardiopulmonary exercise testing of a woman with homozygous ISCU mutations revealed exercise-induced pulmonary vascular dysfunction. Thus, driven by acquired (hypoxia) or genetic causes, the miR-210-ISCU1/2 regulatory axis is a pathogenic lynchpin causing Fe-S deficiency and PH. These findings carry broad translational implications for defining the metabolic origins of PH and potentially other metabolic diseases sharing similar underpinnings. Synopsis Hypoxia-inducible miR-210 down-regulates its targets ISCU1/2 to regulate pulmonary vascular and endothelial levels of Fe-S clusters upon acquired injury or mutations. A patient with ISCU loss-of-function mutation presents with pulmonary vasculopathy. In mouse and human pulmonary vascular and endothelial tissue affected by PH, hypoxic induction of miR-210 and repression of its targets ISCU1/2, down-regulates iron-sulfur levels. Using genetic and pharmacologic methods to perform gain-of-function and loss-of-function analyses of miR-210 and ISCU1/2 in the pulmonary vasculature of mice, the miR-210-ISCU1/2 axis is shown to be necessary and sufficient for induction of hypoxic PH. Cardiopulmonary exercise testing of a woman with ISCU mutations revealed exercise-induced pulmonary vascular dysfunction. Introduction Iron–sulfur (Fe-S) clusters ([4Fe-4S] and [2Fe-2S]) are critical bioinorganic prosthetic groups that are essential for electron transport and consequent metabolic processes (Beinert et al, 1997). The formation of Fe-S clusters is controlled by a conserved set of assembly and scaffold proteins. Current knowledge regarding the importance of these proteins in human disease has been derived largely through investigation of genetic mutations (as reviewed by Rouault, 2012). Yet, stemming from the relatively rare occurrence of these genetic mutations and technical obstacles to measuring these prosthetic groups in mammals, the regulation and actions of Fe-S clusters in the wide spectrum of human metabolic disease have been poorly investigated. Pulmonary hypertension (PH) is a deadly and increasingly prevalent vascular disease where Fe-S biology may figure prominently. PH is defined by increased pulmonary arterial pressure and lung vasculopathy, triggered by varied and often disparate stimuli (Schermuly et al, 2011). Among these triggers, hypoxia and the actions of its master transcription factors of hypoxia, HIF-1α and HIF-2α, are well-recognized insults in multiple PH subtypes, including pulmonary arterial hypertension (PAH, WHO Group 1) (Bonnet et al, 2006; Fijalkowska et al, 2010; Farha et al, 2011; Marsboom et al, 2012) as well as PH associated with hypoxic lung diseases (WHO Group 3) (as reviewed by Tuder et al, 2013). The pulmonary vascular response to hypoxia and HIF factors in PH is incompletely characterized but increasingly has been linked to chronic repression of mitochondrial metabolism (Cottrill & Chan, 2013). Some factors have been identified to modulate metabolic processes in the diseased pulmonary vasculature, but the entire complement of regulators remains undefined. Studying cultured cells, we previously reported that hypoxia up-regulated the HIF-α-dependent microRNA-210 (miR-210), leading to specific mitochondrial and metabolic alterations (Chan et al, 2009). In hypoxia, this microRNA (miRNA) decreased expression of its targets ISCU1 and ISCU2 (iron–sulfur cluster assembly proteins 1/2 or typically described by the single term, ISCU1/2). In mammalian cells, two splice isoforms of ISCU exist and both serve as scaffolding proteins essential for the biogenesis of Fe-S clusters. Both transcripts carry an identical 3′ UTR, but they differ in their location: ISCU1 is located in the cytosol, whereas ISCU2 is located in the mitochondria (Tong & Rouault, 2000). We found that down-regulation of ISCU1/2 decreased Fe-S-dependent mitochondrial respiration and promoted a metabolic shift toward glycolysis for energy production. In the acute setting, this adaptive metabolic shift improved cell survival. However, the effects in health or disease of chronic activation of the miR-210-ISCU1/2 axis with consequent repression of Fe-S clusters in vivo are not known. Based on the pathologic consequences of metabolic dysfunction in other diseases such as cancer, we hypothesized that chronic repression of Fe-S biogenesis directly drives dysfunction of mitochondrial metabolism, cellular proliferation, and frank disease. By interrogating that model further in both acquired injury (e.g. hypoxia) and genetic human disease (ISCU mut/mut), we now define the miR-210-ISCU1/2 regulatory axis as a crucial pathogenic lynchpin of pulmonary vascular disease in vivo. Results The miR-210-ISCU1/2 regulatory axis is activated in PH related to hypoxia and HIF activity Based on our prior studies of the regulation of miR-210 in pulmonary arterial endothelial cells (PAECs) (Chan et al, 2009) and the up-regulation of both HIF-1α and HIF-2α in vascular endothelial cells in hypoxia-relevant PH in mice (Supplementary Fig S1), we hypothesized that the miR-210-ISCU1/2 axis is active in various forms of PH stemming from hypoxia-dependent or HIF-dependent activity. By RT–PCR, we found that miR-210 was induced in the lungs of mice deficient in the von Hippel–Lindau gene (VHL−/−), a genetic model of PH driven by constitutive HIF-α activation (Fig 1A, Supplementary Fig S2A). miR-210 was also up-regulated in lungs of mice suffering from PH stemming from chronic hypoxia (10% O2) (Supplementary Fig S2B) and in mouse lungs with more severe PH (Supplementary Fig S2A) stemming from chronic hypoxia accompanied by serial administration of the VEGF-receptor antagonist SU5416 (Fig 1A). Corresponding with the link between inflammatory cytokine stimulation and up-regulated HIF activity, miR-210 was also induced in inflammatory mouse models of PH driven by transgenic pulmonary interleukin-6 (IL-6) expression [elevated right ventricular systolic pressures reported in Steiner et al (2009)] and by chronic S. mansoni infection (Fig 1A, Supplementary Fig S2A). In correlation, in situ miRNA staining was performed using specific mouse models where reliable histologic assessment of pulmonary vascular cell types was possible. Such staining revealed that miR-210 expression was induced within the diseased pulmonary vasculature of mice (< 100 μm external diameter vessels) (Fig 1B and C), as compared with non-diseased tissue and miR-scrambled control probe (Supplementary Fig S3A). Induction of miR-210 was observed in remodeled pulmonary vessels (Fig 1B) but not in other peripheral vascular tissue (Supplementary Fig S4A). MiR-210 expression was also up-regulated in the small diseased pulmonary arterioles (< 200 μm external diameter) in human PAH lung tissues compared with those arterioles observed in non-diseased human lung tissue (Fig 1D; patient demographics in Supplementary Table S1). Among other vascular cell types, staining for miR-210 was also evident in the intimal layer of remodeled vessels, consistent with prior studies implicating the robust actions of miR-210 in PAECs (Chan et al, 2009). Circulating, extracellular miR-210 was also significantly elevated in plasma sampled adjacent to the pulmonary vascular space (pulmonary capillary wedge position, PCWP) from PH individuals (mean pulmonary arterial pressures, mPAP ≥ 25 mmHg, PCWP ≤ 15 mmHg) as compared with PH-free individuals (mPAP < 25 mmHg, Fig 1E, demographic information in Supplementary Table S2). Such findings are consistent with a growing literature reporting that diseased tissues, which express increased levels of specific miRNAs, can release those miRNAs into the extracellular space and into the circulating plasma (Creemers et al, 2012). Conversely, the direct miR-210 targets ISCU1/2, detected specifically by immunohistochemical stain (Supplementary Fig S3B), were down-regulated in diseased pulmonary vasculature of mice and humans (Fig 1F and G) suffering from PH, but not in unaffected peripheral vasculature (Supplementary Fig S4B–D). Figure 1. The miR-210-ISCU1/2 regulatory axis is activated in endothelium and remodeled vessels in PH A. RT–qPCR reveals that miR-210 was increased in lungs of mice with PH triggered by various conditions: VHL−/− as compared with VHL+/+ mice (N = 4/group), ***P < 0.0001 (first graph); hypoxia + SU5416 (Hyp + SU5416) (N = 6/group) as compared with normoxia + SU5416 (Norm + SU5416) (N = 7/group), **P = 0.0015 (second graph); Il6 transgenic versus littermate control mice (N = 4/group), **P = 0.0097 (third graph); and S. mansoni-infected mice (N = 4) compared with non-infected control mice (N = 5), ***P < 0.0001 (fourth graph). B. From animal subjects in (A), in situ hybridization (ISH, purple stain) revealed increased miR-210 in < 100-μm pulmonary vessels of mice suffering from PH, *P = 0.0493 for first graph, **P = 0.0015 for second graph, *P = 0.0391 for third graph. C. Representative ISH stain of miR-210 in < 100 μm pulmonary vessels of mice (bottom micrographs) exposed to Hyp + SU5416 compared with Norm + SU5416 (α-smooth muscle actin stain from serial sections, top row of micrographs). D. Increased miR-210 in < 200-μm remodeled pulmonary vessels of patients suffering from PAH (N = 19, Supplementary Table S1) as compared with non-PAH donor control lung (N = 10). Serial staining with hematoxylin and eosin is displayed in the top row of micrographs; quantification of miRNA ISH, right graph, *P = 0.0167. E. Increased levels of miR-210 in plasma drawn from the pulmonary circulation (pulmonary capillary wedge position, PCWP) of patients with elevated mean pulmonary arterial pressures (mean PAP ≥ 25 mmHg) compared with control subjects (mPAP < 25 mmHg, N = 5/group, demographics in Supplementary Table S2), *P = 0.0357. F, G. From animals in (C) and humans in (D), immunohistochemistry (IHC) revealed that the miR-210 targets ISCU1/2 were reciprocally down-regulated in miR-210-enriched remodeled pulmonary vessels—namely in PH mice exposed to Hyp + SU5416 (F, ***P = 0.0002) and in human PAH patients (G, ***P = 0.0008). H, I. miR-210 expression (H) was increased (***P = 0.0003), and ISCU1/2 expression (I) was decreased (***P < 0.0001) in PECAM+ pulmonary vascular endothelial cells isolated from PH mice (Hyp +

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